Cells constitute the tissues of our body and are responsible for producing various changes in response to different situations. For instance, the repair of damaged DNA. DNA resides within the cell... Show moreCells constitute the tissues of our body and are responsible for producing various changes in response to different situations. For instance, the repair of damaged DNA. DNA resides within the cell nucleus and can be transcribed and translated into proteins, which play vital roles in numerous cellular processes. The cell relies on modifying existing proteins to carry out essential functions. These modifications can involve the conjugation of small molecules such as Ubiquitin (Ub) or Small Ubiquitin-like Modifiers (SUMOs), leading to protein degradation, conformational changes or intracellular relocation of critical proteins. The conjugation of these small molecules involves a well-orchestrated sequence of enzymatic activities performed by dedicated enzymes: E1 (activating), E2 (conjugating) and E3 (ligase). Among these, the E3 ligase enzymes hold significant importance as they confer substrate specificity.In this thesis, we have developed an advanced Mass-Spectrometry technology called TULIP2 (Targets for Ubiquitin Ligases Identified by Proteomics 2), which facilitates the identification of Ubiquitination targets for specific E3 ligases of interest. Using this technology, we have investigated the BRCA1-BARD1 E3 ligase and explore the in vivo role of the E2 UBE2D3. Furthermore, we have adapted the TULIP2 technology to create the SUMO Activated Target Traps (SATTs), enabling the identification of an E3-specific SUMO proteome. Show less
Post translational modifications (PTMs) are orchestrated by highly active and reversible enzymatic systems to regulate the functional diversity of proteins. Because of their dynamic nature, PTMs... Show morePost translational modifications (PTMs) are orchestrated by highly active and reversible enzymatic systems to regulate the functional diversity of proteins. Because of their dynamic nature, PTMs are used by the cell as a controllable system to regulate a wide variety of processes. Studying modifications of proteins will give us more insight in how the cell uses PTMs to regulate cellular processes and how different PTMs act together to adjust the function of proteins. The research described in this thesis focuses one of these PTMs, the Small ubiquitin-Like Modifier (SUMO). SUMOs are proteins that are covalently attached to lysines in target proteins. These studies have uncovered hundreds of SUMO target proteins and acceptor sites and revealed a role for SUMOylation in protein degradation, cell cycle progression and DNA repair Show less
